4.8 | Intracellular delivery of Sap
Sap was thiolated by reacting it with 2-iminothiolane hydrochloride (25
eq) for 1.5 h on ice[37,38]. PCNDs were suspended
in thiolated Sap solution (2 μM), followed by mechanical rotation in a
refrigerator (4 °C) overnight. After washing, Sap-conjugated PCNDs were
modified with 9E5-NeutrAvidin conjugates, as described above. DLD1 cells
were treated with Sap- and 9E5-conjugated PCND suspension for 3 h.
PCND-treated cells were exposed to ultrasound, as described above. After
ultrasound exposure, the cells were cultured for 48 h and then stained
with calcein-AM. The cells were harvested from the dish using 0.25%
trypsin-EDTA and analyzed using a flow cytometer (Guava easyCyte 8;
Luminex Japan Co. Ltd., Tokyo, Japan).